Hemagglutination by Porphyromonas gingivalis (formerly Bacteroides gingivalis) has been studied for over 15 years as a suspected virulence factor of this species. We have recently cloned two genes, designated ST2 and ST7, from genomic DNA of P. gingivalis 381 that encode proteins which promote hemagglutination. Clone ST2 appears to encode a product of 125 kdal which cross reacts immunologically with 43 and 37 kdal surface- associated proteins of P. gingivalis while the other HA gene, restriction mapping or Southern blot analysis. This proposal is designed to continue the characterization of the P. gingivalis HAs. We propose to complete the determination of the DNA nucleotide sequence of both HA genes and identify the functional domains of the HA molecules. Experiments will also be completed to determine if either of the HA molecules contain proteolytic activity associated with erythrocyte lysis, as has been recently suggested by other researchers. In order to determine the biological activity or function of the HAs, isogenic mutants of each HA gene will be constructed in order to generate "knock-out" mutations in each gene. These constructions will be accomplished by insertional inactivation of the genes by a TcR element into the middle portion of the open reading frame and moving the insertionally inactivated gene "cassette" into a constructed suicide vector which replicates in E. coli but not P. gingivalis. E. coli transformants containing the isogenic construct will then be mated by conjugation with P. gingivalis cells. Only those recipient P. gingivalis in which homologous recombination between the wild-type and inactivated HA genes has occurred should be resistant to tetracycline. All tetracycline resistant colonies will be tested for the presence of defective HA gene by immunological and functional assays. A double mutant containing mutations in both HA genes will also be constructed using a second resistance gene. The isogenic mutant strains will be compared with the wild-type parent strain in vitro adherence assays as well as in in vivo virulence assays in order to determine the function and virulence properties of each gene. Finally, attempts will made to determine the identity of the erythrocyte receptor molecule for each P. gingivalis HA by a variety of approaches including enzymatic treatment of the erythrocytes, fractionation and solubilization of erythrocyte membranes, affinity purification of the solubilized receptor molecule using immobilized HA proteins or whole cells, and liposome technology employing fractionated erythrocyte membranes. It is expected that these studies will result in a complete physical/biochemical characterization of the P. gingivalis HAs, determination of functional/virulence properties of the HAs and information concerning the receptor target molecules on the erythrocyte membrane.